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Whole genome sequence analysis (digital DNA-DNA hybridization and average nucleotide identity) was carried out for 81 sequenced full genomes of the genus Gardnerella, including ten determined in this study, and indicated the existence of 13 genomic species, of which five consist of only one strain and of which only five contain more than four sequenced genomes. Furthermore, a collection of ten Gardnerella strains, representing the emended species G. vaginalis and the newly described species Gardnerella leopoldii, Gardnerella piotii and Gardnerella swidsinskii, was studied. Matrix-assisted laser desorption ionization time-of-flight MS analysis of the protein signatures identified specific peaks that can be used to differentiate these four species. Only strains of G. vaginalis produce β-galactosidase. We emend the description of G. vaginalis (type strain ATCC 14018 T =LMG 7832 T =CCUG 3717 T ) and describe the novel species Gardnerella leopoldii sp. nov. (UGent 06.41 T =LMG 30814 T =CCUG 72425 T ), Gardnerella piotii sp. nov. (UGent 18.01 T =LMG 30818 T =CCUG 72427 T ) and Gardnerella swidsinskii sp. nov. (GS 9838-1 T =LMG 30812 T =CCUG 72429 T ).

We previously reported the presence of an OXA-23 carbapenemase in an undescribed species of the genus Acinetobacter isolated from horse dung at the Faculty of Veterinary Medicine, Ghent University, Belgium. Here we include six strains to corroborate the delineation of this taxon by phenotypic characterization, DNA-DNA hybridization, 16S rRNA gene and rpoB sequence analysis, % G+C determination, MALDI-TOF MS and fatty acid analysis. The nearly complete 16S rRNA gene sequence of strain UG 60467(T) showed the highest similarities with those of the type strains of Acinetobacter bouvetii (98.4 %), Acinetobacter haemolyticus (97.7 %), and Acinetobacter schindleri (97.2 %). The partial rpoB sequence of strain UG 60467(T) showed the highest similarities with 'Acinetobacter bohemicus' ANC 3994 (88.6 %), A. bouvetii NIPH 2281 (88.6 %) and A. schindleri CIP 107287T (87.3 %). Whole-cell MALDI-TOF MS analyses supported the distinctness of the group at the protein level. The predominant fatty acids of strain UG 60467(T) were C12 : 0 3-OH, C12 : 0, C16 : 0, C18 : 1ω9c and summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH). Strains UG 60467(T) and UG 60716 showed a DNA-DNA relatedness of 84 % with each other and a DNA-DNA relatedness with A. schindleri LMG 19576(T) of 17 % and 20 %, respectively. The DNA G+C content of strain UG 60467(T) was 39.6 mol%. The name Acinetobacter gandensis sp. nov. is proposed for the novel taxon. The type strain is UG 60467(T) ( = ANC 4275(T) = LMG 27960(T) = DSM 28097(T))., (© 2014 IUMS.)

A Gram-stain-positive, obligately anaerobic, short rod, designated strain HHRM1715(T), was isolated from the blood of a patient with Fournier's gangrene, complicated by sepsis. On the basis of 16S rRNA gene sequence analysis, strain HHRM1715(T) was shown to belong to the genus Atopobium and was most closely related to Atopobium minutum (95 % similarity). The results of 16S rRNA-gene-based phylogenetic analysis, cellular fatty acid analysis and differential biochemical tests, showed that strain HHRM1715(T) represented a novel species of the genus Atopobium. We therefore describe Atopobium deltae sp. nov. with HHRM1715(T) ( = LMG 27987(T) = CCUG 65171(T)) as the type strain and propose an emended description of the genus Atopobium with regard to the DNA G+C content., (© 2014 IUMS.)

Three Gram-negative strains, NF 1078(T), NF 1598 and NF 1715, were isolated from clinical (two) and environmental (one) samples, respectively. Sequence analysis of the 16S rRNA genes revealed similarity of 100% among the three strains and next highest similarity to the type strain of Acidovorax avenae (98.16%). The three strains were able to acidify lactose and rhamnose on low peptone phenol red agar and to alkalinize citrate on Simmons' agar and were negative for nitrate reduction. The DNA G+C content of strain NF 1078(T) was 67.1 mol%. The level of DNA-DNA relatedness between this strain and the type strains of A. avenae (ATCC 19860(T), LMG 2117(T)) was 29%. Based on these phylogenetic, phenotypic and genotypic analyses, the three strains could be distinguished clearly from all other recognized Acidovorax species and should be classified as representatives of a novel species of the genus Acidovorax, for which the name Acidovorax wautersii sp. nov. is proposed. The type strain is NF 1078(T) (=LMG 26971(T)=CCUG 62584(T)).

Two clinical strains, NF 296 and NF 931, present in our collection, were identified biochemically as members of CDC group II-i. Determination of the 16S rRNA gene sequence revealed highest similarity with strains of Sphingobacterium mizutaii. Because these strains produced indole, whereas S. mizutaii has been described as indole-negative, we also investigated the type strain and a reference strain of S. mizutaii, LMG 8340(T) (=CCUG 15907(T)) and LMG 8341 (=CCUG 15908), and found both strains also to be positive for indole production. These data warrant inclusion of some of the CDC group II-i strains into S. mizutaii and emended descriptions of Sphingobacterium mizutaii as indole-production-positive and of the genus Sphingobacterium as variable for indole production.

Human Psychrobacter isolates, other than Psychrobacter phenylpyruvicus, are predominantly designated Psychrobacter immobilis. Phenotypic and genotypic testing of Psychrobacter isolates that have been deposited in different culture collections as P. immobilis indicates that most of these human isolates belong to the species Psychrobacter faecalis and Psychrobacter pulmonis.

Acinetobacter genospecies (genomic species) 10 and 11 were described by Bouvet and Grimont in 1986 on the basis of DNA-DNA reassociation studies and comprehensive phenotypic analysis. In the present study, the names Acinetobacter bereziniae sp. nov. and Acinetobacter guillouiae sp. nov., respectively, are proposed for these genomic species based on the congruence of results of polyphasic analysis of 33 strains (16 and 17 strains of genomic species 10 and 11, respectively). All strains were investigated by selective restriction fragment amplification (i.e. AFLP) analysis rpoB sequence analysis, amplified rDNA restriction analysis and tDNA intergenic length polymorphism analysis, and their nutritional and physiological properties were determined. Subsets of the strains were studied by 16S rRNA gene sequence analysis and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS or had been classified previously by DNA-DNA reassociation. Results indicate that A. bereziniae and A. guillouiae represent two phenetically and phylogenetically distinct groups within the genus Acinetobacter. Based on the comparative analysis of housekeeping genes (16S rRNA and rpoB genes), these species together represent a monophyletic branch within the genus. Despite their overall phenotypic similarity, the ability to oxidize d-glucose and to grow at 38 degrees C can be used in the presumptive differentiation of these two species from each other: with the exception of three strains that were positive for only one test, A. bereziniae strains were positive for both tests, whereas A. guillouiae strains were negative in these tests. The strains of A. bereziniae originated mainly from human clinical specimens, whereas A. guillouiae strains were isolated from different environmental sources in addition to human specimens. The type strain of A. bereziniae sp. nov. is LMG 1003(T) (=CIP 70.12(T) =ATCC 17924(T)) and that of A. guillouiae sp. nov. is LMG 988(T) (=CIP 63.46( T) =ATCC 11171(T) =CCUG 2491(T)).

A total of 95 isolates, belonging to 33 species of five dermatophyte genera, i.e. Arthroderma (15 species), Chrysosporium (two), Epidermophyton (one), Microsporum (three) and Trichophyton (12), were studied using internal transcribed spacer 2 (ITS2)-PCR-RFLP analysis (ITS2-RFLP), consisting of amplification of the ITS2 region, restriction digestion with BstUI (CG/CG) and restriction fragment length determination by capillary electrophoresis. ITS2-RFLP analysis proved to be most useful for identification of species of the genera Arthroderma, Chrysosporium and Epidermophyton, but could not distinguish between several Trichophyton species. The identification results are in agreement with established and recent taxonomical insights into the dermatophytes; for example, highly related species also had closely related and sometimes difficult-to-discriminate ITS2-RFLP patterns. In some cases, several ITS2-RFLP groups could be distinguished within species, again mostly in agreement with the taxonomic delineations of subspecies and/or genomovars, confirming the relevance of ITS2-RFLP analysis as an identification technique and as a useful taxonomic approach.

Many species of non-fermenting Gram-negative bacilli (non-fermenters) are important opportunistic and nosocomial pathogens. Identification of most species of non-fermenters by phenotypic characteristics can be difficult. In this study, an oligonucleotide array was developed to identify 38 species of clinically relevant non-fermenters. The method consisted of PCR-based amplification of 16S-23S rRNA gene intergenic spacer (ITS) regions using bacterial universal primers, followed by hybridization of the digoxigenin-labelled PCR products with oligonucleotide probes immobilized on a nylon membrane. A total of 398 strains, comprising 276 target strains (i.e. strains belonging to the 38 species to be identified) and 122 non-target strains (i.e. strains not included in the array), were analysed by the array. Four target strains (three reference strains and one clinical isolate) produced discrepant identification by array hybridization. Three of the four discordant strains were found to be correctly identified by the array, as confirmed by sequencing of the ITS and 16S rRNA genes, with the remaining one being an unidentified species. The sensitivity and specificity of the array for identification of non-fermenters were 100 and 96.7%, respectively. In summary, the oligonucleotide array described here offers a very reliable method for identification of clinically relevant non-fermenters, with results being available within one working day.

A collection of eight clinical strains from Belgian hospitals and three clinical strains of the CCUG collection were characterized biochemically as being similar to CDC groups II-h and II-c; the latter differs from group II-h only by positivity for sucrose acidification. These 11 strains were found to cluster according to 16S rRNA gene sequence similarity at a level of >or=99.5%, and on the basis of their tDNA-PCR profile. Based on 16S rRNA gene sequence analysis, this collection of strains was related most closely to Chryseobacterium hispanicum (97.2%), but they differed from the type strain of this species by the following phenotypic characteristics: growth at 37 degrees C, negativity for xylose acidification, positivity for acetate assimilation-alkalinization on Simmons' agar base and absence of flexirubin pigments, and by their tDNA-PCR profile. Strain NF802T showed only 57.8% DNA-DNA relatedness to the type strain of C. hispanicum. Fatty acid composition did not enable differentiation from C. hispanicum. The DNA G+C content of strain NF802T is 36.5 mol%. The name Chryseobacterium hominis sp. nov. is proposed for this taxon, with type strain NF802T (=CCUG 52711T=CIP 109415T).

The feasibility of sequence analysis of the ribosomal 16S-23S intergenic spacer region (ITS) was evaluated for identification of 24 species of Streptococcus, one species of Abiotrophia, 18 species of Enterococcus and three species of Granulicatella. As GenBank currently lacks ITS sequence entries for many species of these four genera, the ITS sequences of 38 type strains were first sequenced and submitted to GenBank to facilitate species identification of these genera. Subsequently, the ITS sequences of 217 strains (84 reference strains and 133 clinical isolates) were determined and species identification was made by blast search for homologous sequences in public databases. Species other than Streptococcus contained multiple ITS fragments and only the shortest fragment was analysed. A total of 25 isolates (11.5 %) produced discrepant identification by ITS sequencing. The 25 discordant strains were analysed further by sequencing of the 16S rRNA gene for species clarification, and 21 were found to be identified correctly by ITS sequence analysis. The correct identification rate by ITS sequencing was 98.2 % (213/217). Several closely related enterococcal and streptococcal species/subspecies contained specific ITS signature sequences that were useful for differentiating these bacteria. In conclusion, ITS sequencing provides a useful approach towards identifying this group of pathogens on a molecular platform alongside 16S rRNA gene sequencing.

A total of 26 isolates of non-fermenting, Gram-negative rods, obtained between 1980 and 2004 by various clinical laboratories in Belgium, with phenotypic characteristics resembling those of members of the genera Chryseobacterium and Empedobacter (indole-positive) and a biochemical profile resembling that of CDC group II-h, but urease-positive, were collected at the Université Catholique de Louvain Microbiology Laboratory, Belgium. The 16S rRNA gene sequences were determined for most of the isolates and showed 94-95 % similarity with the type strain of Empedobacter brevis as the closest relative, indicating that these isolates might belong to a separate genus. Furthermore, the 16S rRNA gene sequences of the isolates were similar, but two clusters (genomovars) could be distinguished. The sequence similarities were 99.5-100 % for the 14 isolates of genomovar 1 and 99.4-100 % for the 12 isolates of genomovar 2. The similarity between the two clusters was 98.3-99.5 %. The presence of two clearly different groups was corroborated by using tRNA intergenic length polymorphism analysis, which also enabled differentiation of the novel species from all other species studied thus far using this technique. DNA-DNA hybridization results excluded a close relatedness to Empedobacter brevis. The DNA G+C contents of the reference strains of genomovars 1 and 2 were 33.8+/-0.4 and 34.4+/-0.2 mol%, respectively. The name Wautersiella falsenii gen. nov., sp. nov., is proposed for this group, comprising two closely related genomovars. The type strain of the species and reference strain for genomovar 1 is NF 993(T) (=CCUG 51536(T)=CIP 108861(T)), which was isolated from a surgical wound. The reference strain for genomovar 2 is NF 770 (=CCUG 51537=CIP 108860), which was isolated from blood.

In the present study, it was shown that repetitive-DNA-element PCR fingerprinting using the (GTG)5 primer [(GTG)5-PCR] is a rapid and reliable tool to genotypically differentiate members of the recently described pan-European multi-resistant Acinetobacter baumannii (MAB) clone III from the known MAB clones I and II. The identification of four new representatives of the MAB clone III dating from 1991 to 1993 by (GTG)5-PCR indicates that this clone has persisted in European hospitals since the beginning of the 1990s. Tetracycline (TET) resistance was found to be common among clone III strains, including one strain that also displayed resistance to minocycline. The TET-resistance phenotype in this MAB clone appeared to be strongly associated with the presence of the efflux-type gene tet(A), but the fact that some members lack this gene or have acquired an additional tet gene [i.e. tet(M)] suggests that the tet gene carriage in the pan-European clone III population may have diversified in time and space. In contrast, all clone III strains shared the previously described aminoglycoside resistotype encoded by the aminoglycoside-modifying genes aphA6 and the class 1 integron-associated aadB, which may point to the fact that these genes probably are more stably inherited in MAB clone III compared to tet genes.

Four staphylococcal isolates from clinical and necropsy specimens from a cat, a dog, a horse and a parrot (Psittacus erithacus timneh) were found to constitute a distinct taxon. 16S rRNA gene sequence analysis revealed that its closest phylogenetic relatives are Staphylococcus intermedius and Staphylococcus delphini. Growth characteristics, biochemical features and DNA-DNA hybridizations demonstrated that the strains differ from these and other known species and that they represent a single, novel Staphylococcus species for which the name Staphylococcus pseudintermedius sp. nov. is proposed. The novel species is commonly confused with S. intermedius in routine diagnostic veterinary bacteriology. Although the strains described were isolated from lesions and show several characteristics typical of pathogenic staphylococci, such as coagulase, DNase and beta-haemolysin production, the pathogenic significance of the novel species remains unclear. The type strain, LMG 22219(T) (=ON 86(T)=CCUG 49543(T)), was isolated from lung tissue of a cat.

Comparative 16S rDNA sequence analysis indicates that two distinct sublineages, with a sequence dissimilarity of >4 % (bootstrap value, 100 %), exist within the genus RALSTONIA: the Ralstonia eutropha lineage, which comprises Ralstonia basilensis, Ralstonia campinensis, R. eutropha, Ralstonia gilardii, Ralstonia metallidurans, Ralstonia oxalatica, Ralstonia paucula, Ralstonia respiraculi and Ralstonia taiwanensis; and the Ralstonia pickettii lineage, which comprises Ralstonia insidiosa, Ralstonia mannitolilytica, R. pickettii, Ralstonia solanacearum and Ralstonia syzygii comb. nov. (previously Pseudomonas syzygii). This phylogenetic discrimination is supported by phenotypic differences. Members of the R. eutropha lineage have peritrichous flagella, do not produce acids from glucose and are susceptible to colistin, in contrast to members of the R. pickettii lineage, which have one or more polar flagella, produce acid from several carbohydrates and are colistin-resistant. Members of the R. pickettii lineage are viable for up to 6 days on tryptic soy agar at 25 degrees C, whereas members of the R. eutropha lineage are viable for longer than 9 days. It is proposed that species of the R. eutropha lineage should be classified in a novel genus, Wautersia gen. nov. Finally, based on the literature and new DNA-DNA hybridization data, it is proposed that Pseudomonas syzygii should be renamed Ralstonia syzygii comb. nov.

The taxonomic status of seven glucose-non-acidifying, non-proteolytic Acinetobacter strains characterized by forming small colonies on agar media was studied. With one exception, all strains were from human specimens. They could be distinguished from all described Acinetobacter (genomic) species by their ability to grow on ethanol and acetate as sole sources of carbon but not on 22 other substrates tested including DL-lactate or DL-4-aminobutyrate. DNA-DNA hybridization studies, 16S rRNA gene sequence analysis, amplified rDNA restriction analysis and DNA polymorphism analysis by AFLP showed that these strains represent a hitherto unknown species of the genus Acinetobacter, for which the name Acinetobacter parvus (type strain LMG 21765(T)=LUH 4616(T)=NIPH 384(T)=CCM 7030(T)) is proposed.

Three clusters of isolates have previously been defined within the species Comamonas terrigena, on the basis of DNA-rRNA and DNA-DNA hybridization data, and of protein electrophoretic patterns and immunotyping. More detailed characterization in the current study shows that representatives of these three groups can also be differentiated phenotypically from each other. Strains of C. terrigena sensu stricto (C. terrigena DNA group 1) are pyrrolidone aminopeptidase-positive, do not grow at 40 degrees C, are L-alanine-positive and are always negative for 4-hydroxybenzoate. Strains of C. terrigena DNA groups 2 and 3 are pyrrolidone aminopeptidase-negative; the former is the only group that is tyrosine-negative, and only the latter can grow at 42 degrees C (with an optimal growth temperature of 40 degrees C). These findings are corroborated by differences in 16S rDNA sequence and tRNA intergenic spacer lengths. Therefore, it is proposed to rename C. terrigena DNA group 2 [containing former Aquaspirillum aquaticum and E. Falsen (EF) group 10 strains] as Comamonas aquatica sp. nov., and C. terrigena DNA group 3 (containing former EF group 10 strains) as Comamonas kerstersii sp. nov.

Streptococcus ferus is a mutans-like streptococcus originally isolated from wild rats fed with sugar cane. Taxonomically, this species has not been studied extensively. Ten Gram-positive coccal strains, isolated from the tonsils and nasal conchae of piglets, were identified as S. ferus by 16S rDNA sequencing, tRNA-intergenic spacer length polymorphism analysis (tDNA-PCR), whole-cell protein profiling using SDS-PAGE, G+C content determination and extensive biochemical testing. In all these tests, the type strain of S. ferus (LMG 16520T), from a rat, was included. The results of the tests are described and an emended species description is presented.